Cell experiment:

To evaluate the effect of Aconine on the viability of RAW264.7 cells, cytotoxicity assays are performed using the Cell Counting Kit-8. Briefly, the cells are seeded in 96-well plates at a density of 2×104, 3×103, 1.2×103, 1×103 or 1×103 cells/well in the presence or absence of Aconine (0.125-0.5 mM) for 8 h, 24 h, 48 h, 5 d or 7 d, respectively. After incubating the cells with CCK-8 solution for 2 h, optical density is measured at 450 nm using a GENios microplate reader. Cell viability is expressed as a percentage of the control[1].

Aconine inhibits receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced NF-κB activation.

Treatment with Aconine significantly inhibits the RANKL-induced transcriptional activity of NF-κB in a dose-dependent manner. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP. Aconine (0.125, 0.25 μM) does not affect the viability of RAW264.7 cells, but dose-dependently inhibits RANKL-induced osteoclast formation and bone resorptive activity. Aconine dose-dependently inhibits the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduces the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which plays an important role in cell-cell fusion[1].

References:
[1]. Zeng XZ, et al. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression. Acta Pharmacol Sin. 2016 Feb;37(2):255-63.